An amperometric enzyme-channeling immunosensor.

This paper presents a new disposable amperometric, enzyme-channeling immunosensor for a quantitative, rapid, separation-free enzyme immunoassay (EIA) that can be used in clinical diagnostics, as well as in biomedical, biochemical, and environmental research. The sensor consists of a disposable, polymer-modified, carbon electrode on which enzyme 1 is coimmobilized with a specific antibody that binds the corresponding antigen in a test solution. The solution also contains a conjugate of enzyme 2. An immunological reaction brings the two enzymes into close proximity at the electrode surface, and the signal is amplified through enzyme channeling. The localization of both enzymes on the electrode surface limits the enzymatic reactions to the polymer/membrane/electrode interface. The sensor overcomes the problem of discriminating between the signal that is produced by the immuno-bound enzyme label on the electrode surface and the background level of signal that emerges from the bulk solution. Combining enzyme-channeling reactions, optimizing hydrodynamic conditions, and electrochemically regenerating mediators within the membrane layer of the antibody electrode significantly increased the signal-to-noise ratio of the sensor. The amperometric enzyme-channeling immunosensor enabled the performance of separation-free EIAs without washing steps, resulting in a relatively short assay time of 5-30 min for the complete immunoassay, compared with at least 1-3 h for ELISA methods. Model systems using peroxidase-antibody, biotin-avidin, viral antigens (CD4-gp120), and bacteria (Staphylococcus aureus) were investigated. S. aureus cells were detected in pure culture at concentrations as low as 1000 cells/ml.